Fungi for improvements of wood and pulp appearance and qualities

ABSTRACT

The invention relates to the use of fungi stains of  Ophiostoma floccosum, Ophiostoma piceae  or  Ophiostoma pluruanulatum , or mixtures on wood and pulp to improve chemical pulping processes and/or reduce cooking tie and/or improve brightness and/or decrease extractives.

TECHNICAL BACKGROUND

[0001] The present invention relates to the use of certain fungi in theenhancement of wood quality. More particularly, but not exclusively, itrelates to the use of particular fungi strains of Ophiosotoma floccosum,Ophiostoma piceae or Ophiostoma pluruanulatum, or mixtures of strains,to elicit a useful effect on wood or wood products derived therefrom inor on a substrate or locum e.g. including biocontrol; the prevention ofstaining of cellulosic materials from detrimental strains or otherdetrimental effects from detrimental strains, or for the reduction ofpitch components and/or their detrimental effects, the production ofpositive effects achieved from the strains in regard to solid wood withuptake of liquid materials including but not limited to glue,preservative, varnish, paint, improvement in mechanical or chemicalpulping as achieved by increased efficiency of cooking liquor (withconcomitant lower kappa number value achieved for the equivalent cookingliquor or less cooking liquor required for the same kappa numberachieved without use of strains); and increased steam penetrationefficiency. The kappa value is an indication of the amount of bleachingchemical required to remove the residual lignin.

BACKGROUND ART

[0002] Wood is a complex material composed of cellulose, hemicellulose,lignin, and wood extractives or a resinous material commonly calledpitch. “Resin” or “pitch” (used interchangeably) includes that complexmixture of hydrophobic substances in wood, which are soluble in neutralorganic solvents such as methylene chloride, diethyl ether, benzylalcohol and the like. These include the terpenes, the diterpene(“resin”) acids, fatty acids and esters, glycerides and waxes as well asalcohols, hydrocarbons and other compounds associated therewith.

[0003] In the production of products from wood pulps, the presence ofpitch is undesirable as it frequently forms deposits which are difficultto remove or its presence causes detrimental properties to the productsderived from the wood.

[0004] Another problem in the timber industry derives from staining.When trees are cut down they commonly become infected by any one or moreof a variety of fungi which can stain the wood in any one or more of avariety of colours. A major problem in the industry today involves lossof value in timber products, and any products derived therefrom due tothe unsightly staining caused by so-called blue stain fungi which cancolour the wood a variety of colours including gray, dark blue andblack, such staining appearing in the wood even though the outersurfaces or regions of the wood have been cut away in forming thelumber.

[0005] In the early 1990's workers at Sandoz Chemicals Biotech ResearchCorporation and their colleagues at the University of Minnesotadeveloped a resin degradative and biocontrol fungus for pretreatment ofwood from an Ophiostoma species. This work resulted in the developmentof the current commercial product, the fungal inocula Catapip®97. It wasmade by classical mating of isolated ascospores from various Ophiostomapiliferum isolates of the United States. Ophiostoma piliferum is asaprophytic Ascomycete found throughout the world, and commonly referredto as one of the sapstain fungi.

[0006] Sapstain fungi grow mainly in wood in the ray parenchyma cells,within resin canals, within tracheids and fibre cells and penetratesimple and bordered pits, occasionally forming bore holes through woodcell walls. Sapstain fungi are not capable of degrading cellulose orlignin, but metabolise resin extractives, starch and simple sugars.Sapstain fungi cause a characteristic stain of sapwood resulting in ablue, black, grey or brown discolouration of the wood, and isresponsible for major economic losses in the timber and some pulpingindustries. Problems with sapstain are most prevalent in warm, humidclimates and when wood with high sapwood content is used (Kay et al,1997).

[0007] Ascospore mating and selection procedures on loblolly pineproduced the isolate Cartapip®97 which grew rapidly on wood chips,degraded substantial quantities of pitch and did not stain wood (Farrellet al, 1992; Wendler et al, 1992; Hoffmann et al, 1992; Farrell et al,1993). This colourless (albino) isolate was shown to be blocked in the1,8-dihydroxynaphthalene (DHN) melanin synthetic pathway by theinability to produce the intermediate scytalone (Zimmerman et al 1993).Later work showed that O. piceae albino strains could also be generatedby the same methodology (White-McDougall 1998). Treatments of wood chipswith the albino fungi resulted in a biocontrol effect, such that thealbino fungus quick grew throughout the chips and suppressed the growthof staining fungi (Farrell et al, 1993). Biocontrol of sapstain in logsusing the Cartapip 97 inoculum was demonstrated on red pine in the laband in th field (Benhrendt et al 1995; Behrendt et al, 1995).

DISCLOSURE OF THE INVENTION

[0008] It is an object of the present invention to provide an inoculumand a process which enhances wood quality, having advantages over thosepreviously known, to provide a process which enhances wood quality ofwood (including but not limited to New Zealand woods or woods of theSouther Hemisphere), or to at least provide the public with a usefulalternative.

[0009] Our recent studies dealing with New Zealand sapstaining organismshave yielded surprising results. Studies particularly with radiata pinehave shown certain New Zealand Ophiostoma albino species especiallyalbino or very slightly pigmented in their hyphae; even if slightlypigmented in other fungal bodies provide improved results in biocontrolof sapstain and reduction of pitch. These species have shown to providean improved result over that of Cartapip®97 on radiata pine. Cartapip®97has proved to be somewhat but not fully effective to reduce stain to thedesired industry target of <10% stain in 3 months. Furthermore, ourstudies have shown mixtures of more than one albino fungus have alsobeen effective in increasing brightness of wood, and/or brightnessincrease in chemical pulp resulting from albino treatment of wood,and/or improved pulping efficiency such that lower kappa numbers wereachieved with treatment of the wood with he albino strains.

[0010] As used hereinafter reference to specific stains means thefollowing:

[0011]Ophiosotoma floccosum strain F13—that strain deposited as AGALaccession number NM00/12246 on 16/08/00

[0012]Ophiosotoma floccosum strain F40—that strain deposited as AGALaccession number NM00/12247 on 16/08/00

[0013]Ophiosotoma floccosum strain F71—that strain deposited as AGALaccession number NM00/12490 on 16/08/00

[0014]Ophiosotoma floccosum strain F80—that strain deposited as AGALaccession number NM00/12491 on 16/08/00

[0015]Ophiasotoma floccosum strain F93—that strain deposited as AGALaccession number NM00/12492 on 16/08/00

[0016]Ophiosotoma floccosum strain F3410—that strain deposited as AGALaccession number NM00/12252 on 16/08/00

[0017]Ophiosotoma floccosum strain F5040—that strain deposited as AGALaccession number NM00/12251 on 16/08/00

[0018]Ophiosotoma floccosum strain F4650—that strain deposited as AGALaccession number NM00/12253 on 16/08/00

[0019]Ophiosotoma floccosum strain F7073—that strain deposited as AGALaccession number NM00/12493 on 16/08/00

[0020]Ophiostoma piceae strain OPC 422—that strain deposited as AGALaccession number NM00/12248 on 16/08/00

[0021]Ophiostoma piceae strain OPC 580—that strain deposited as AGALaccession number NM00/12249 on 16/08/00

[0022]Ophiostoma piceae strain OPC 194—that strain deposited as AGALaccession number NM00/12250 on 16/08/00

[0023]Ophiostoma piceae strain OPC 703—that strain deposited as AGALaccession number NM00/12489 on 16/08/00

[0024] In a first aspect of the present invention there is provided aninoculum for wood comprising or including one or more (preferablybiologically pure) forms of a fungal culture of the species Ophiostomafloccosum and/or Ophiostoma piceae and/or Ophiosotoma pluruanulatumeffective to reduce the amount of colour staining caused by woodstaining fungi.

[0025] Preferably said fungal culture is selected from the followingstrains:

[0026]Ophiosotoma floccosum F71,

[0027]Ophiosotoma floccosum F80,

[0028]Ophiosotoma floccosum F93,

[0029]Ophiosotoma floccosum F40,

[0030]Ophiosotoma floccosum F13,

[0031]Ophiostoma piceae OPC 703,

[0032]Ophiostoma piceae OPC 422,

[0033]Ophiostoma piceae OPC 580,

[0034]Ophiostoma piceae OPC 194,

[0035]Ophiostoma pluruanulatum 3410,

[0036]Ophiostoma pluruanulatum 7073,

[0037]Ophiostoma pluruanulatum 5040,

[0038]Ophiostoma pluruanulatum 4650

[0039] In one embodiment the wood source may be wood chips and thefungus or fungi may be applied by spraying the wood chips. In analternative embodiment the wood source may be logs to be cut intostructural wood, and inoculation includes inoculating at least one endof the logs, and preferably all around the logs' surfaces. In a furtheralternative embodiment the wood source may be structural wood andinoculation includes inoculating at least 60% of the surface area of thestructural wood. In another alternative embodiment the wood source maybe structural wood and inoculation includes inoculating at least 60% ofthe surface area of the Structural wood, which may then be later madeinto chips or a subsequent fibre product.

[0040] Preferably the wood to be inoculated may be a conifer such as butnot limited to Radiata pine, Douglas fir or Loblolly pine, and it mayalso be a hardwood including but not limited to eucalyptus, oak, poplar,or aspen.

[0041] In a second aspect of the invention the is provided abiologically pure culture of a strain of Ophiostoma floccosum having allof the identifying characteristics of one of the fungi of strains F40,F13, F71, F80 and F93.

[0042] In a third aspect of the invention there is provided a biologicalpure culture of a strain of Ophiostoma piceae having all of theidentifying characteristics of one of the fungi of strains OPC 703, OPC422, OPC 580 and OPC 194.

[0043] In a fourth aspect of the invention there is provided abiologically pure culture of a strain of Ophiostoma pluruanulatum havingall of the identifying characteristics of one of the fungi of strains3410, 7073, 5040 and 4650.

[0044] In a fifth aspect of the invention there is provided a method ofreducing the amount of colour staining caused by wood staining fungi inand/or on a wood source which comprises or includes inoculating at leasta portion of the wood source with an effective amount of at least onefungus selected from the group consisting of the species Ophiosotomafloccosum, Ophiosotoma piceae and Ophiostoma pluruanulatum, which iscapable of reducing the amount of colour staining caused by the woodstaining fungi.

[0045] Preferably said fungi may be selected from the following stains:

[0046]Ophiosotoma floccosum F13,

[0047]Ophiosotoma floccosum F40,

[0048]Ophiosotoma floccosum F71,

[0049]Ophiosotoma floccosum F80,

[0050]Ophiosotoma floccosum F93,

[0051]Ophiostoma piceae OPC 703,

[0052]Ophiostoma piceae OPC 422,

[0053]Ophiostoma piceae OPC 580,

[0054]Ophiostoma piceae OPC 194,

[0055]Ophiostoma pluruanulatum 3410,

[0056]Ophiostoma pluruanulatum 7073,

[0057]Ophiostoma pluruanulatum 5040,

[0058]Ophiostoma pluruanulatum 4650

[0059] In one embodiment the wood source may be wood chips and thefungus or fungi may be applied by spraying the wood chips. In analternative embodiment the wood source may be logs to be cut intostructural wood, and inoculation includes inoculating at least one endof the logs, and preferably all around the logs' surfaces. In a furtheralternative embodiment the wood source may be structural wood andinoculation includes inoculating at least 60% of the surface area of thestructural wood.

[0060] Preferably the wood source may derive from a conifer such as butnot limited to, Radiata pine, Douglas fir or Loblolly pine and it mayalso be a hardwood including but not limited to eucalyptus, oak, poplar,or aspen.

[0061] In a sixth aspect of the present invention there is provided aninoculum for wood comprising or including one or more (preferablybiologically pure) forms of a fungal culture of the species Ophiostomafloccosum, and/or Ophiostoma piceae and/or Ophiosotoma pluruanulatumeffective in reducing the pitch content of wood.

[0062] Preferably said fling may be selected from the following strains:

[0063]Ophiosotoma floccosum F40

[0064]Ophiosotoma floccosum F13

[0065]Ophiostoma piceae OPC 422

[0066]Ophiostoma piceae OPC 580

[0067]Ophiostoma piceae OPC 194

[0068] In one embodiment of the wood source may be wood chips and thefungus or fungi may be applied by spraying the wood chips. In analternative embodiment the wood source may be logs to be cut intostructural wood, and inoculation includes inoculating at least one endof the logs. In a further alternative embodiment the wood source may bestructural wood and inoculation includes inoculating at least 60% of thesurface area of the structural wood.

[0069] Preferably the wood to be inoculated may be but is not limited toa conifer such as Radiata pine, Douglas fir or Loblolly pine and it mayalso be a hardwood including but not limited to eucalyptus, oak, poplar,or aspen.

[0070] In a seventh aspect of the invention there is provided abiologically pure culture of a strain of Ophiostoma floccosum having allof the identifying characteristics of one of the fungi of strains F40and F13.

[0071] In an eighth aspect of the invention there is provided abiologically pure culture of a strain of Ophiostoma piceae having all ofthe identifying characteristics of one of the fungi of strains OPC 422,OPC 580, OPC 194.

[0072] In a ninth aspect of the invention there is provided a method forreducing the pitch content in a wood source and/or improving brightnessin chemical pulp resulting from the albino treatment of wood, and/orimproving pulping efficiency such that lower kappa numbers were achievedwith treatment of the wood with the albino strains, which comprises orincludes inoculating at least a portion of the wood source with aneffective amount of at least one fungus selected from the groupconsisting of the species Ophiosotoma floccosum, Ophiosotoma piceae andOphiostoma pluruanulatum, which is capable of reducing the pitchcontent.

[0073] Preferably said fungi may be selected from the following strains.

[0074]Ophiosotoma floccosum F40

[0075]Ophiosotoma floccosum F13

[0076]Ophiostoma piceae OPC 422

[0077]Ophiostoma piceae OPC 580

[0078]Ophiostoma piceae OPC 194

[0079] Preferably the method may further include the step of maintainingthe inoculated wood source under conditions which allow fungal growthfrom the inoculation for a term sufficient to effect a reduction of thepitch content of the wood source by such inoculated fungal growth.

[0080] In one embodiment the wood source may be pulpwood, orunsterilised pulpwood, or unsterilised refined pulpwood. Alternativelythe wood source may be wood chips and the fungus or fungi is applied byspraying the wood chips. Alternatively the wood source may be debarkedor undebarked timber or logs.

[0081] Preferably the wood source may derive from but not limited to aconifer such as Radiata pine, Douglas fir or Loblolly pine and it mayalso be a hardwood including but not limited to eucalyptus, oak, poplar,or aspen.

[0082] Preferably the method may include using or applying more than onefungal strain.

[0083] In a tenth aspect of the invention there is provided a biologicalpure culture of a strain of Ophiostoma floccosum having all of theidentifying characteristics of one of the fungi of AGAL AccessionNumbers NM00/12246, NM00/12247, NM00/12490, NM00/112491 or NM00/12492.

[0084] In an eleventh aspect of the invention there is provided abiologically pure culture of a strain of Ophiostoma piceae having all ofthe identifying characteristics of one of the fungi of AGAL AccessionNumbers NM00/12248, NM00/12249, NM00/12250 or NM00/12489.

[0085] In a twelfth aspect of the invention there is provided abiologically pure culture of a strain of Ophiostoma pluruanulatum havingall of the identifying characteristics of one of the fungi of AGALAccession Numbers NM00/1225, NM00/12252, NM00/12253 or NM00/12493.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE

[0086] From the 21 sapstain fungal species isolated and identified inNew Zealand (Farrell et al, 1998), four species were chosen for abreeding programme in order to make albinos/white fungi. These NewZealand albinos were made as had been done with Cartapip 97, classicallymating a variety of isolates of the same species to produce albino,non-melanising strains. Neither genetic engineering nor mutagens wereused to make the isolates. The albino isolates were studied for theirability to biocontrol and reduce staining in mature radiata pine logs(Farrell et al 1997a; Farrell et at 1997b), and for their ability toreduce resin components in radiata pine wood chips.

[0087] Surprisingly and a significant advancement over what is presentlycommercially available, were the effect of some of the Ophiostomafloccosum, O. pluruanulatum, and O. Piceae albinos. In this examplethese stains were isolated from New Zealand but it is expected thatthese species and their subsequent albinos could prove beneficial whenisolated from any country and origin and give beneficial effectssuperior to Cartapip®97, Some of the New Zealand albinos weresignificantly better at maintaining biocontrol effect and exclusion ofdetrimental organisms, including stain fungi, and degrading detrimentalwood components than others and better than the commercial product. Alsosurprising and unobvious according to presently granted patents was thefact that different albinos of various species had differing degrees ofeffect, with some of the albinos providing other than a positive effecton wood with their application and incubation.

FIGURES

[0088]FIG. 1: Results of solid logs with albino Inoculation.

[0089]FIG. 2: 80,000 ton trial—Extractives of chips as a function ofincubation time in the chip piles.

[0090]FIG. 3: Chips inoculated with both OF40 and OF13.

Example 1

[0091] Competition Assays

[0092] Albinos were constructed by single ascospore isolation andmating, according to Zimmerman, W. C., Blanchette, R. A., Burnes, T. A.,Farrell, R. L. (1993). Melanin and perithecial development in Ophiostomapiliferum. Mycologia 87: 857-863.

[0093] Albinos were tested in the laboratory for presence of pigmentunder a variety of incubating conditions and in competition assays. Thecompetition assays were conducted by inoculating either sterilised,gamma irradiated or some other method, or non-sterilised radiata pinecubes or chips with each or more than one of sapstaining species,including but not limited to Sphaeroides sapinea, Ophiostoma piliferumor Leptographium species, prior to, simultaneously or after inoculationwith the albino or more than one albino fungus. In most assays thestaining fungus/fungi was introduced to the chips as actively growingvegetative cells at one-tenth the cell density as the albino(s), anextreme situation with respect to challenge.

[0094] An example below shows typical conducted lab competition assays,which consisted of albinos made from New Zealand ascomycetes, and thecommercial product Cartapip®897. TABLE 1 Intensity Comments F71 0 F71 +Stainer same day 0.8 successful F71 + Stainer next day 0.5 successfulF73 0 F73 + Stainer same day 1 successful F73 + Stainer next day 1.3 Notsuccessful F74 0.5 Not successful F74 + Stainer same day 1.5 Notsuccessful F74 + Stainer next day 1.5 Not successful F77 0 F77 + Stainersame day 2 Not successful F77 + Stainer next day 0.5 successful F80 0.5Successful F80 + Stainer same day 1 Successful F80 + Stainer next day 1Successful F81 0 Successful F81 + Stainer same day 2 Not SuccessfulF81 + Stainer next day 2 Not Successful F87 0 Successful F87 + Stainersame day 1.2 Not successful F87 + Stainer next day 1.5 Not successfulF93 0 Successful F93 + Stainer same day 0.3 successful F93 + Stainernext day 0.5 successful OPC703 0.2 Not successful OPC703 + Stainer sameday 0.3 successful OPC703 + Stainer next day 0.7 successful OPC422 0.5Not successful OPC422 + Stainer same day 0.8 successful OPC422 + Stainernext day 1 successful Intensity % coverage OPC194 0 successful OPC194 +Stainer same day 3 Not successful OPC194 + Stainer next day 3 Notsuccessful OPC580 0 successful OPC580 + Stainer same day 2 Notsuccessful OPC580 + Stainer next day 1.8 Not successful 3410 0Successful 3410 + Stainer same day 0.2 Successful Intensity Comments3410 + Stainer next day 0.5 Successful 7073 0 Successful 7073 + Stainersame day 0.5 Successful 7073 + Stainer next day 0.7 Successful Cartapip97 0.2 Not successful Cartapip + Stainer same day 0.7 successfulCartapip + Stainer next day 1.7 Not successful Stainer same day 3.0Stainer next day 3.3 Uninoculated sterile control 0

[0095] Albino concentration was 10⁶ blastospore/ml

[0096] Stainers (fungi which cause stain including but not limited toLeptographium, or Diplodia (Sphaeropsis sapinea) or any other organismthat leaves a sin on wood) concentration was 10⁶ blastospore/ml

Example 1

[0097] Results

[0098] Surprisingly not all of the albinos had the same effect forprevention of stain. This was an unexpected and unobvious result aspreviously it was considered that any albino or white fungus, or almostalbino would constitute a biocontrol effect on wood, and preventstaining fungi from staining the wood, Successful competitive albinofungi were judged as those with less than 0.5 stain score on their own,and that maintained a stain score when challenged with one of thestaining fungi, in this example Leptographium or Diplodia (Sphaeropsissapinea) of 1 or less than 1. As shown in the results, the preferredorganisms were F71, F80, F93 (all albinos, with regard to hyphae but notwith regard to synnemata, of Ophiosotoma floccosum), OPC 703, 422(albinos, with regard to hyphae but not with regard to synnemata, of thespecies Ophiostoma piceae) and 3410 and 7073 (albinos, with regard tohyphae but not with regard to synnemata, of the species Ophiostomapluruanulatum). The commercial Product Cartapip®97. had too much stainand was not successful.

Example 2

[0099] In order to teach the mechanism of positive effect upon wood, thealbino fungi have been tested in the laboratory for growthcharacteristics and enzyme production, By standard biochemistry assaysthe albino fungi have been tested for their ability to deplete starch ingrowth media, and units of activity in media of amylase and lipaseenzymes. TABLE 2 Albino Characteristic Chart Key Characteristics of NewZealand Ophiostoma albinos: Nutrient depletion Cube Competition Albino %Starch Amylase Liplo O.pilaf Growth in Species ID # Depletion/hr LipaseLepto(L) (D) (Op) Blocks pluruanulanim 3410 92/24 700 342 3440 98/24 3645040 2226 486 4630 1620 4650 540 4680 1119 673 4880 738 4890 88/24 4135980 97/24 576 7052 100/24 840 643 1 1 1 7058 92/24 917 701 1 1 1 706085/100 843 566 1 1 1 7073 721 1 1 1 7076 98/48 1527 384 1 1 1 7078 10412 1 floccosum F3 74 1 1 P F13 293 1 1 P F29 270 496 2 1 P F36 44/24 226653 1 1 P F61 92/24 230 526 P F63 21/24 235 365 P F30 45/24 388 482 1 1P F20 16/24 502 562 1 1 1 P F35 100/48 959 604 1 1 P F40 17/24 462 728 11 1 P piccae 1 156 piccae 194 14/24 180 442 P 422 25/24 177 421 1 P 54213/24 1091 782 1 1 P 400 95/24 1055 702 P 12 553 1 P 580 11/24 960 614 2P

[0100] Stain during competition is marked on a scale of 1-5 with 0 beingwhite wood and 5 black stain. Each competitive experiment is markedindividually.

Example 2

[0101] Results

[0102] There is a variety of responses of the albino fungi to thesediagnostic tests. Not all the albinos have the same characteristics.

Example 3

[0103] Field Results on Solid Logs with Albino Inoculation

[0104] Albino fungi were grown in liquid cultures of growth mediaconsisting of sufficient carbon, nitrogen and trace elements andvitamins to promote cell biomass accumulation (standard and non-standardmycological media are acceptable), either used in the spent growthmedia, or harvested by centrifugation and resuspended in water orbuffer, with or without washing, were sprayed onto radiata pine logs,Either a pure single strain or more than one strain can be usedsimultaneously or consecutively. The typical cell concentration of thefungus in spent media, water or buffer was from 10e2 to 10e6 colonyforming units per millilitre. The fungal cell suspensions were appliedonto the logs by back-pack spray systems, or commercial sprayers, eithervariety such as used by commercial agricultural spray systems orcommercial log spray systems. Fungal cell suspensions could also beapplied to logs by dipping the logs into a bath suspension containingthe cells in water, buffer or spent growth media. Fungal application canbe done only one time or more than one time. Mouldicides may be usedafter a few days of the fungal application to improve surfaceappearance. After application of the fungal cell suspension to the logs,the logs were let in the field, or in an enclosed container such aswarehouse, boat etc, and after 1, 2, 3 or longer months the amount of inwas assessed on the logs visually. Results of one such application ofmultiple fungi are given in FIG. 1.

Example 3

[0105] Results

[0106] Some albinos can have a positive effect on wood quality,biocontrol and keep wood from staining, better than the commercialfungus and better than commercial anti-sapstain chemicals. Some albinoshave detrimental effects and increase the amount of stain, therefore notall white fungi behave in the same manner with regard to their effect onwood.

EXAMPLE 4

[0107] Resin Component Degradation

[0108] Solid wood or wood chips, either previously sterilised by gammairradiation or some other method, or non-sterilised, was treated withmore than two dozen albino fungi, a bacteria (for example Pseudomonasresinovorans) or the commercial product Cartapip 97. The inoculated woodwas either left to stand at ambient temperature or incubated from 0degrees Centigrade to plus 65 degrees Centigrade. The wood was analysedanytime from 2 days to 16 weeks, most typically between 1-8 weeks) forfull extractive analysis.

[0109] Extractives Analysis

[0110] The wood extractives content of treated and untreated chips wasdetermined on freeze dried chip samples which had been ground to <0.5mm. The wood meal was extracted by solvent extraction with acetone usinga Soxtec extractor. Gas chromatography of the methylated extract wasperformed using a DB-1 5 m×0.32 mm capillary column fitted with a 0.5m×0.5 mm deactivated silica retention gap and using an on columninjection technique. Detection was by flame ionisation detector andquantitation was performed using the internal standard technique on thechromatography data system.

Example 4

[0111] Results

[0112] Control, non-fugal inoculated radiata pine wood chips wereanalysed as a reference for resin acids, fatty acids and glycerides, andthe results are given in Table 3, TABLE 3 Analysis of Wood Extractivesof Control Chips from Lab Experiment Sample % Acetone % Free % Resin %Control 1.26 0.227 0.411 0.130 Control 1.27 0.156 0.282 0.081 Average:1.26 0.19 0.35 0.11

[0113] Results for the fungal treated samples can be most easilycompared as to the effect of one fungal isolate as compared to anotherfungal, rather than compared to the reference control, non-fungaltreated samples.

[0114] Dozens of albino isolates were treated for their ability in thelaboratory to degrade wood extractives. Results given in Table 2 show induplicate some of the better and the lesser fungal overall effects forwood extractives decrease. TABLE 4 Analysis of Wood Extractives afterFungal Treatment % % Free Acetone Fatty % Resin Sample Extractives AcidsAcids % Glycerides OPC 542 1.46 0.092 0.730 0.025 ″ 1.70 0.096 0.7970.018 F63 1.19 0.108 0.481 0.020 ″ 1.02 0.080 0.375 0.021 7060 0.730.039 0.226 0.012 ″ 0.74 0.040 0.222 0.015 7073 0.72 0.073 0.184 0.039 ″0.68 0.069 0.168 0.036 7076 1.19 0.120 0.268 0.093 ″ 1.15 0.120 0.2650.089 OPC 422 0.57 0.044 0.133 0.033 ″ 0.61 0.048 0.150 0.038 F13 0.620.036 0.100 0.016 ″ 0.64 0.036 0.095 0.017 OPC 580 0.65 0.038 0.1650.010 ″ 0.70 0.050 0.217 0.015 F40 0.37 0.028 0.129 0.017 ″ 0.58 0.0430.156 0.023

[0115] As shown in Table 4, the various isolates tested on wood chipsvaried considerably in their ability to degrade wood extractives. Therange of extractives values for differing fungal treatments was from0.37% to 1.70% by weight of wood; obviously some much more efficient atdegrading the wood extractives than others. Fungi such as the isolate O.floccosum F40 on the average decreased total acetone extractives to0.47% versus OPC 542, with the same length of time incubating on thechips, had an average acetone extractives value of 1.58% by weight ofwood. Analogously, some of the fungi had significant decreases in freefatty acids, for example O. floccosum F40 with free fatty acids of0.035% and free resin acids of 0.143% whereas O. floccosum isolate F63did not decrease the free acids as much with average % free fatty acidsof 0.094% and % free resin acids of 0.428% by weight of wood.

[0116] The free fatty acids and free resin acids were further analysedby gas chromatography. Examples of the data for isolate F13 are given inTable 5.

[0117] All the data obtained to date on wood extractive decrease wassorted for the possible contenders for further evaluation on the basisthat their resin acid contents were below 0.2% by weight of wood; Theseisolates were O. floccosum F40 and F13, and O. piceae C OPC 422, OPC580, and OPC 194. TABLE 5 Analysis of Free acids and extractivecomponents after F13 Lab Fungal Treatment-duplicate analysis %Extractives 0.62 0.64 mg Analysed 0.546 0.574 C16:0 found (ug) 4.76 4.96C18:2 found (ug) 5.94 5.51 C18:1 found (ug) 19.88 20.53 C18:0 found (ug)4.01 4.09 Free Fatty Acids Sum (ug) 34.59 35.09 % Free Fatty Acids inExtract 6.34 6.11 Corrected % FFA in Extract 5.82 5.60 % Free FattyAcids in Sample 0.039 0.039 Corrected % FFA in Sample 0.036 0.036Pimaric found (ug) 15.31 14.73 Sandara. found (ug) 3.19 3.13 Isopimaricfound (ug) 8.34 7.59 Levo. + Pal. found (ug) 16.53 16.02 Dehydro. found(ug) 44.26 42.96 Abietic found (ug) 2.94 3.04 Neoabietic found (ug) 5.815.59 Resin Acids Total (ug) 96.38 93.06 % Resin Acids in Extract 17.6516.21 Corrected % RA in Extract 16.20 14.85 % Resin Acids in Sample0.109 0.104 Corrected % RA in Sample 0.100 0.095 C16:0 found (ug) 3Unidentified (ug) 14.62 16.47 C18:1 found (ug) C18:2 found (ug) C18:0found (ug) Glycerides Sum (ug) 14.62 16.47 % Glycerides in Extract 2.682.87 Corrected % Glyc. in 2.52 2.68 % Glycerides in Sample 0.017 0.018Corrected % Glyc. in 0.016 0.017 % Extract Identified 24.5 23.1

Example 5

[0118] Effect of Albinos on Processed Pulp

[0119] Fungal cell cultures as described in Example 1 were appliedeither to logs or to chips at a ratio of 1000 gallons cell suspension inwater to 200 tons wet weight chips—this cell suspension to wood weightratio could be varied by several orders of magnitude.

[0120] In this Example are given the results from the use of two O.floccosum albinos mixed together and applied onto chips, or three O.piceae albino fungi mixed together and sprayed onto wood chips. Controlpile of chips sprayed just with water were also established.

[0121] OF Pile: in this example consisted of chips sprayed with waterplus a mixture of two Ophiostoma albinos, O. floccosum 40, and O.floccosum 13, but other combination of albino fungi could also be used.OPC Pile in this example consisted of chips sprayed with water plusmixture of three O. piceae C albino isolates, OPC 580, OPC 422 and OPC194 cell concentrate, but other combination of albino fungi could alsobe used.

[0122] Sprayed in 4500 litre water solution onto 200 tons wet weightchips. Temperature or rain/moisture does not affect the spray trials.

[0123] Materials and Methods

[0124] Survey, Culture Isolations and Identification, and CompetitionExperiments.

[0125] Sampling, isolations, identifications, mating, culture growth andcompetition experiments on wood cubes and chips were done as describedby Farrell, et al, 1998

[0126] Analysis of the Mill Trial

[0127] Spray Inocula for the Mill trial

[0128] The O. piceae 540, O. piceae 422, O. piceae 194 culturesuspensions were mixed together and applied to one pile, and O.floccosum 40 and O. floccosum 13 were mixed together and applied toanother pile, as described in the Materials and Methods and details inAppendix 2. From the spray tank, a sample of the inocula was taken atstart middle and end of the spraying of the 200-ton wet weight of chips,to verify the mixing and dosage of the blastospores per volume spray wasconsistent throughout the trial.

[0129] Fungal Growth during Eight Week Trial

[0130] Growth of the fungi at the mill piles was monitored by standardmycological culturing of chips taken from the four piles every two weeksduring the trial and with many isolates checking with molecular markersfor DNA analysis. Consistently, the albino O. floccosum grew from the OFpile. There was little if any growth from the OPC pile of O. piceae andthere was O. floccosum, growing in this pile, the native O. floccosum ofthe site The Wet Pile showed positive Ophiostoma identification ofnative O. quercus, O. floccosum Rhizopus; Trichoderma, Fusarium andyeasts. Therefore the Tantanoola Mill site consistently has the presenceof moulds, yeasts and three sapstain organisms O. quercus, O. floccosumand Graphium species C on the radiata pine wood chips.

[0131] Analysis of the Pulps of the Mill Trial

[0132] DNA: The wood chips of the piles were made into pulp at the millby normal procedures. Samples of pulp were taken for further analysisfrom the Chemiwasher, Twin wire wash press (TWWP), and bleached pulp(M57). DNA extractions were performed on the pulps to show whether therewas any trace of the New Zealand-origin fungi; these analyses showedthat there was no residual DNA left in the pulps. Polymerase chainreaction (PCR) of specific DNA probes for O. piceae and O. foccosum onthe extractions confirmed that no DNA could be isolated from the pulps,and hence no PCR reaction was observed; therefore there was no residualDNA in the pulps from the applied albino fungi.

[0133] EXTRACTIONS: Soxtec dichloromethane extractions of the pulps fromthe chips of the 8 week mill trial (Dry pile not processed) werecompared to the mill data from May 1999 to February 2000, with seasoningof chips routinely during this period being 12-16 weeks and results aregiven in Table 6. TABLE 6 Wood Extractives of Pulps from Mill (averagevalues based on wood weight) SampleChemiwasherTWWPM57Mill data May'99-Feb 2000 0.90% 0.10% 0.07% OF Pile pulp 0.92 0.12% 0.05% OPC Pilepulp 0.90% 0.12% 0.06% Wet Pile pulp 0.88% 0.10% not done

[0134] TWWP=Twin wire wash press

[0135] Interestingly, as evident in the data of Table 6, it can beobserved for commercial utility that the amounts of wood extractivesafter fungal treatment (OF Pile Pulp or OPC Pile Pulp) did not decreaserelative to the Wet Pile pulp, nor from the normal mill wood extractivesof pulp data as collected from May '99-February 2000. Therefore,contrary to the laboratory results of the fungi (Example 4 showing resincomponent degradation by the organisms of choice), in the true settingof the utility of the fungi the resin component numbers are notdecreased from the controls or normal mill running without fungal use.

[0136] BRIGHTNESS: Brightness's of the pulps from the fungal treatedpulps showed that the OPC pile pulp from the TWWP had 54.0% ISObrightness, and after bleaching 78.3% ISO brightness. The OF pile, whichduring the growth assays showed significant growth of the albino O.floccosum, and less growth of staining species showed improvedbrightness with the OF pip from the TWWP having a brightness of 66.8%ISO brightness and after bleaching a 85.5% ISO brightness, a significantimprovement in brightness. TABLE 7 BLEACHING RESULTS: O. floccosum O.piceae Mill seasoning Caustic 66.8% 54.0%   59% extracted Bleached 85.5%78.3% 80-82%

[0137] Finally a further experiment comprising inoculating 6000 tons offungi with O. floccosum 13 and 40 was performed. In processing the pulpthrough chemical pulping after the fungal incubation, the mill againexperienced an upward brightness shift of approximately 5 points. Thepulp was processed through the mill without any problems.

[0138] The other unexpected benefit which became clear during theextended trial was a Kappa shift downwards of 5 points. This result mayresult in a huge cost saving as it means the mill can do more for lesswith regard to the cooking liquor charge on wood. Cooking liquor is oneof the major manufacturing expenses. This benefit of the 5 pointdownwards shift of kappa (the kappa test is made from the reaction ofpulp with acidic permanganate solution and the calculated numberresulting is indicative of the amounts of non-cellulosic components(especially lignin) residing in the pulp, used in mill control work toindicate the degree of delignification occurring during cooking and thechemical requirement for bleaching (G. A. Smook, Handbook for Pulp &Paper Technologists)) to pulp mills is a significant achievement ofincreased chemical pulping efficiency. The increased pulping efficiencyhas been suggested in a research publication (M B Wall, G Stafford, YNoel, A Fritz, S Iverson, R Farrell, Treatment with O. piliferumImproves Chemical Pulping Efficiency, presented at the 6th internationalConference an Biotechnology in the Pulp and Paper Industry, Vienna, Jun.11-15, 1995), but this publication showed in the laboratory only adownward shift of 1.25 kappa points and this work has never beendemonstrated in a mill, and in a true commercial setting. The increasedchemical pulping efficiency described here has practical significance tomills in several ways, including but not limited to, decreased amount ofcooking liquor to be added to fungal treated wood chips to reachequivalent kappa (lignin) content at the same amount of cellulosiccontent, and as measured at the same viscosity, greater yield of pulpfrom equivalent cooking liquor as the cooking liquor works with greaterefficiency to remove lignin and not cellulosic contents, improved pulpand fibre properties as the reduced cooking liquor does not act as muchnon-specifically on cellulosic components, improved benefit to theenvironment because less cooking liquor, and less concomitant effluent,to be used, and reduced cooking time as the cooking liquor is workingwith greater efficiency on fungal treated wood chips.

Example 5

[0139] Results

[0140] The albino O. floccosum sprayed wood chips produced a brighterpulp than had been observed in the mill, and which required less bleachchemical to reach a higher brightness than the standard mill pulps.

[0141] The extractives analysis indicated that the O. floccosum pulps,after 8 weeks incubation, had less extractives than normal seasoningwithout sprayed fungus requiring 12-16 weeks.

Example 6

[0142] 80,000 ton wood chip experiment Our fungal of New ZealandOphiostoma floccosum OF40 and OF13 were researched in this 80,000 tonwood chip trial. The fungal inocula were provided as freeze dried powderin vacuum-sealed bags, packaged as approximately 1000 tonnes wood chipsinocula per bag of each fungus strain.

[0143] A day of inoculation was typically considered about 000 tonneswood chips. [tonne refers to 1,000 kg or 2200 lb. The wood chips usedfor trial purposes were all green wood with no additional water addedprior to inoculation.]

[0144] The trial was established as follows:

[0145] Stage 1: OF 40 sprayed 10,000 tons (10 days)

[0146] Stage 2: OF 40 & OF 13 for 60 days, about 60,000 tons, and

[0147] Stage 3: OF 13 at the end sprayed onto 7000 tonnes,

[0148] Specifics of the chips and the spraying inocula were as follows:

[0149] Average chip size=25 mm×25 mm×2 mm

[0150] Density of wood=350-550 (average 400 kg/cub meter) (400 microgram/cub.millimeter)

[0151] In order to estimate in the future for inocula-loadingefficiency, one chip had a surface area of 1450 square. mm, volume of1250 cub. mm and a mass of 5 gram.

[0152] 1 tonne of wood occupies 2.5 cub.meters, & chipped has an averagesurface area of 290 squ.meter. Fifty tonne of wood was sprayed with 2cub. meters Water/Fungi

[0153] i.e. 2 cubic meters of water is sprayed over an average surfacearea of 14500 squ.meters.

[0154] i.e. 1 cubic meters of water is sprayed over an average surfacearea of 7250 squ.meters.

[0155] i.e. 1 cubic centimetre is sprayed over an average surface areaof 7250 squ.milimeters.

[0156] Since there were about 10,000 (10E+4) to (10E+5) colony formingunits of fungi per millilitre, each square millimetre of chip surfacewas sprayed with 0.725 to 7.25 fungi spores.

[0157] Results of the Trial

[0158] Mill Specifics

[0159] During the trial the mill was constantly making observationsregarding performance of the fungal inoculated chips. A successfuloutcome of the trial was decided at the beginning to have no pitchshutdowns when fungal inoculated chips were run through the mill. Thiswas the case. There were no shutdowns at the mill because of pitchduring the three months of fungal use.

[0160] Importantly, the pulp was clean leaving the mill.

[0161] Analysis Method for Fungal Trial Extractives

[0162] The samples were freeze dried and then ground on a Wiley mill topass a 40 mesh sieve and then extracted using the Soxtec apparatus witht-buthymethyl ether as solvent. The total extractives are expressed onthe basis of oven dried starting wood.

[0163] Gas Chromatography

[0164] (According to Procedure of Orsa and Holmbom, J. Pulp Pap. Sci.,20(12) J361, 1994)

[0165] The internal standard and synthetic mixture solutions wereprepared. The internal standard (50 μL) was added to the extract vialcontaining approximately 0.6 mg of material, and then treated with anexcess of ethereal diazomethane. The solvent and excess diazomethanewere evaporated under a stream of nitrogen dichloromethane (400 μL)added and the vial was sealed.

[0166] A Hewlett Packard 5890 series II GC with on-column injection andFID detector was used, with 1 μL injected onto the column, which was aBP1, 10 m×0.54 mm×2 mm.

[0167] GC Conditions

[0168] Injector temperature=80° C. initially, held for 2 minutes andramped to 340° C. at 20° C./minute and then held at 340° C. Detectortemperature 340° C.

[0169] Oven temperature=40° C. initially, held for 2 minutes. Then itwas increased to 100° C. at 60° C./minute, held for 2 minutes, ramped to200° C. at 4° C./minute and then ramped to 340° C. at 5° C./min and heldfor 10 minutes.

[0170] The helium carrier gas flow rate was constant at 12 kPa.

[0171] All retention times were determined by comparison to authenticstandards.

[0172] Soxtec Method

[0173] The finely ground wood (˜2 g) was accurately weighed in duplicateinto small cellulose thimbles (26 mm×60 mn). At the same time sample (˜1g) was accurately weighed in duplicate for moisture content. The thimblewas put into the Soxtec apparatus and 60 mL t-butylmethyl ether added toa pre-weighed aluminium cup containing anti bumping granules. Thethimble was placed in the boiling position for 30 minutes and thenraised to the rinsing position for 60 minutes. After this time, thesolvent was removed by evaporation, and the last trace of solventremoved under vacuum. The dry cup was re-weighed and the % extractcalculated.

[0174] GC Chromatography

[0175] (a) Extract Preparation

[0176] The extract was washed into a volumetric flask with small volumesof acetone, and made up to the mark. An aliquot was removed to yieldapproximately 0.6 mg of material and transferred to a vial. The acetonewas removed by a gentle stream of nitrogen, and the vial flushed withnitrogen prior to sealing. The vials were stored sealed under nitrogenin the freezer before analysis.

[0177] (b) Solutions

[0178] The internal standard solution contained pentadecanoic acid(0.8472 mg/mL), betulin (0.7782 mg/mL), cholesteryl heptadecanoate (0.94mg/mL) and dipalmitoyloleoyl glycerol (0.4154 mg/mL) in dichloromethane.The pentadecanoic acid is a check for complete derivitisation, and thedipalmitoyloleoyl glycerol is a check for triglyceride recovery. Thebetulin is used as internal standard for resin and fatty acids and thecholesteryl heptadecanoate is used as internal standard fortriglycerides. The ratio of the 2 internal standards is used to monitorcolumn performance.

[0179] A synthetic resin mixture, containing 3 fatty acids, 2 resinacids and a triglyceride was also prepared in dichloromethane to checkresponse factors.

[0180] Percentage component is determined by adding together the areasof peaks of the gas chromatagram that fall within a retention timerange. This range is determined by analysis of authentic standards.However, there is no guarantee that peaks in the range are the same asthe standards. Some verification work has been done on GCMS (gaschromatography/mass spectroscopy) to check peak assignments, howeverthis was not done for triglycerides, where the samples can not be put onthe GCMS. The pitch and pulp samples especially may not actually containtriglycerides at all.

[0181] The Mill sent samples of chips every two weeks of the trial toThe University of Waikato for analysis. These chips had done thefollowing:

[0182] Chips were assayed for the type of fungi growing on them by theWaikato lab's standard mycological procedures and assessed for stains anindication of the albino fungal dominance over other microorganisms onthe chips and biocontrol effect.

[0183] Chips were analysed for extractives.

[0184] Fungal Isolation from Chips

[0185] The fungal isolates from the chips used were isolated andpurified on selected media, and identified by morphology and in somecases by DNA probe. The purpose of these isolations is first, to detectwhether the inoculated fungi sprayed onto the chips were growing, andsecond, to see what else might be growing on the chips. The two albinoO. floccosum strains, called OF 40 and OF13, had a reversion of melaninformation such that when growing on chips, these fungi produced lightbrown synnema hairs that stick up from the chip. The hyphae of the fungiare non-melanised i.e. non-staining. The brown synnema proved useful forthe mill and lab to be able to see when the albino was well growing onthe chip. The data given in Table 9 is from all the research milltrials. TABLE 9 Identification of Fungi from Chips Isolates from 800tonnes wood chips Mill Expt. (Nov '1999) Piles: Wet Pile (water), DryPile (not sprayed), O. piceae pile and O.floccosum pile No Site From ID1 Wet pile O. querci 2 Wet pile O. querci 3 Dry pile Mucor 4 Wet pileGraphium sp. C 5 Wet pile Graphium sp. C 6 Wet pile O. querci 7 O.piceae O. floccosum/Graphium sp. C pile 8-31 O. floccosum O. floccosumalbino O. floccosum/Graphium sp. C, pile O. querci, Pesotum sp. C and Y

[0186] Isolates from 6000 tonnes wood chips Mill Expt. (March 2000).Pile sprayed with 50:50 mixture of O. floccosum 13 and O. floccosum 40No ID 1A Mucor 2B O. floccosum albino 3C Mucor 4D O. floccosum albino 5EO. floccosum 6F O. floccosum 7G O. floccosum 2a O. querci 2c O. querci2d Mucor 2e O. querci 7f O. floccosum Pesotum sp Y

[0187] F40 Treatment

[0188] 0 week: Chips were clean, no stained chips observed.

[0189] The microorganisms that grew on media 4 and 6 after 3-6 daysincubation are: A,niger, Trichoderma, Penicillium, White fungi,Bacteria, Yeast. The albino O40 with brown synnema (O. floccosum)dominated on plates media 6.

[0190] 2 week: Chips were clean, no stain, Albino O40 with Brown synnemacan be seen under microscope/naked eyes.

[0191] After 3-6 day incubation on media 4/6: Mucor, Bacteria, andAlbino O.floccosum (Brown synnema) dominated on media 6.

[0192] 4 week and 6 week; chips were clean no stain at 4 weeks, but at 6weeks some stained chips observed; After 3-6 day incubation of 4/6 weeksamples : Mucor (fungus with an obvious Black dot), Bacteria,Trichoderma, Albino O. floccosum (brown synnema) and darker O. floccosumdominated on media 6.

[0193] 8 and 10 week samples: Chips little bit darker, Albino O.floccosum brown synnema fungi growing well and black dot fungi can beseen under microscope After 3-6 day incubation on media 4/6: Mucor,Rhizopus, bacteria yeast and albino O. floccosum were dominated on media6.

[0194] F13+F40 Treatment

[0195] 2 week: Chips were clean no stain. Brown synnema on chips can beseen under microscope. After 3-6 days on media 4/6, Mucor, Rhizopus,Bacteria and Albino brown synnema (O. floccosum) on media 6.

[0196] 4 and 6 Week Sample:

[0197] 4 week sample still clean, no stained chips, but on 6 week samplechips become darker. Brown synnema and black hairy fungi cm be seenunder microscope.

[0198] On media 4/6: Mucor, Trichoderma bacteria yellowish hypae fungi,albino O. floccosum and darker O. floccosum.

[0199] 8 and 10 week. Some chips were darkened. Black dot fungi on chipsand albino O. floccosum growing well.

[0200] After 3-6 days incubation on media 4/6. Mucor (black dot),Bacteria, Rhizophus. Albino O. floccosum dominated.

[0201] F13 Treatment

[0202] 2 week: Clean, but stain on some chips. Under microscope: blackhairy fungi, Trichoderma, and albino brown synnema (O. floccosum).

[0203] Under microscope observation there were no difference betweensample from surface, and bottom of pile.

[0204] After 3-6 days: Mucor, Aspergillus, brown synnema (floccosum).

[0205] 4 week: Chips look stained. Brown synnema on all chips, Blacksynnema, Black hairy fungi, Trichoderma, Penicillium.

[0206] After 3-6 days: Mucor, bacteria, Trichoderma. Floccosum(dominated).

[0207] 6 and 8 week sample: Chips were darkened and stained, underscope,: Brown synnema (floccosum), Trichoderma, black hairy fungi.

[0208] After 3-6 days: Mucor, bacteria, Trichoderma, Penicillium. Brownsynnema/floccosum dominated.

[0209] 10 week sample: Less stain than samples 6 and 8 weeks. Undermicroscope observe the brown synnema on chips, after 6-8 days on media 4and 6: Penicillium, Mucor, yeast, bacteria, Albino brown synnema (O.floccosum), black synnema (O. querci?)

[0210] Untreated Chips Sample

[0211] 30 week Stock pile.

[0212] Mostly clean no stain, but some chips were colonised with blackhairy fungi.

[0213] 30 wk stock pile: Trichoderma, penicillium, Bacteria, Blacksynnema (Querci).

[0214] Chips: Black synnema (querci?), blach hairy fungi, Penicillium,Mucor, bacteria

[0215] Truck: Penicillium, Mucor, Yeast.

[0216] Analysis

[0217] Statistics Data from Lab and Trial Extractives Data

[0218] Tables A to L present statistical data.

[0219] Statistic extractives for Lab Trial with the parameters Strains:1=F13 2=F40

[0220] Doses 1=0(control), 2={fraction (1/10)}×, 3=×, 4=10×

[0221] Media 1=YM 2=Peptone TABLE A Analysis of Variance for % MTBE(Two-way analysis of Variance between Strain vs doses) Source DF SS MS FP Strain 1 0.3180 0.3180 4.26 0.050 Doses 3 2.1335 0.7112 9.52  0.000**Interaction 3 0.1972 0.0657 0.88 0.465 Error 24  1.7922 0.0747 Total 31 4.4409

[0222] TABLE A Analysis of Variance for % MTBE (Two-way analysis ofVariance between Strain Vs Media) Source DF SS MS F P Strain 1 0.3180.318 2.30 0.140 Media 1 0.250 0.250 1.81 0.189 Interaction 1 0.0030.003 0.02 0.884 Error 28  3.870 0.138 Total 31  4.441

[0223] TABLE C Analysis of Variance for % MTBE (Two-way analysis ofVariance between Doses vs Media) Source DF SS MS F P Strain 3 2.13350.7112 9.96  0.000** Doses 1 0.2503 0.2503 3.50 0.073 Interaction 30.3426 0.1142 1.60 0.216 Error 24  1.7144 0.0714 Total 31  4.4409

[0224] TABLE D Analysis of Variance for % Fatty Acids (Two-way analysisbetween Doses and Media) Source DF SS MS F P Dose 3 0.1959 0.0653 2.070.131 Media 1 0.0968 0.968  3.06 0.093 Interaction 3 0.1080 0.0360 1.140.353 Error 24  0.7587 0.0316 Total 31  1.1594

[0225] TABLE E Analysis of Variance for % FA (Two-way analysis betweenStrain Vs Doses) Source DF SS MS F P Strain 1 0.0242 0.0242 0.70 0.412Media 3 0.1959 0.0653 1.88 0.160 Interaction 3 0.1056 0.0352 1.01 0.404Error 24  0.8336 0.0347 Total 31  1.1594

[0226] TABLE F Analysis of Variance for % Fatty acids (Two-way analysisbetween Strain vs doses) Source DF SS MS F P Strain 1 0.242 0.0242 0.700.412 Doses 3 0.1959 0.653 1.88 0.160 Interaction 3 0.1056 0.352 1.010.404 Error 24  0.8336 0.347 Total 31  1.1594

[0227] TABLE G Analysis of variance for % Resin Acids (Two-way analysisbetween Strain and Doses) Source DF SS MS F P Strain 1 0.0957 0.09573.60 0.70* Doses 3 0.5150 0.1717 6.45  0.02** Interaction 3 0.07920.0264 0.99 0.413 Error 24  0.6386 0.0266 Total 31  1.3286

[0228] TABLE H Analysis of Variance for % Resin Acids (Two-way analysisbetween Strain Vs Doses) Source DF SS MS F P Strain 1 0.0957 0.0957 2.230.147 Doses 1 0.0205 0.0205 0.48 0.495 Interaction 1 0.0088 0.0088 0.200.655 Error 28  1.2036 0.0430 Total 31  1.3286

[0229] TABLE I Analysis of variance for % Resin Acids (Two-way analysisbetween Doses Vs Media) Source DF SS MS F P Doses 3 0.5150 0.1717 7.370.001  Media 1 0.0205 0.0205 0.88 0.357** Interaction 3 0.2343 0.07813.35 0.036*  Error 24  0.5587 0.0233 Total 31  1.3286

[0230] TABLE J Analysis of variance of % Triglycerides (Two-way analysisbetween Doses Vs Media) Source DF SS MS F P Doses 3 0.048838 0.01627952.09 0.000** Media 1 0.005512 0.005512 17.64 0.000** Interaction 30.007937 0.002646 8.47 0.001** Error 24  0.007500 0.000313 Total 31 0.069788

[0231] TABLE K Analysis of variance of % Triglycerides (Two-way analysisbetween Strain and Media) Source DF SS MS F P Strain 1 0.00245 0.002451.11 0.300 Media 1 0.00551 0.00551 2.50 0.125 Interaction 1 0.000200.00020 0.09 0.765 Error 28  0.06163 0.00220 Total 31  0.06979

[0232] TABLE L Analysis of variance of % Triglycerides (Two-way analysisbetween Strain vs Doses) Source DF SS MS F P Strain  1 0.002450 0.0024504.01 0.057* Doses  3 0.048838 0.016279 26.67 0.000** Interaction  30.003850 0.001283 2.10 0.126 Error 24 0.014650 0.000610 Total 310.069788

[0233] Definitions and Explanations

[0234] When varying the dose of the fungi, the mill trial fungal dosewas called 1× (or one times) and in the lab this was varied to one-tenththe mill dose ({fraction (1/10)}×) or 10 times the mill dose (10×).

[0235] % MTBE means the total extractives as removed by methyltert-butyl ether (MTBE) from the wood chips. The components of fattyacids, resin acids and triglycerides were identified on the gaschromatogram. In Table 1, for the Lab data, the terms “Peptone” and “YM”are the growth medium that the lab uses to prepare the fungi. The term“powder” means the freeze-dried powder inocula of the fungi that wasprovided to the mill for the 80,000 tonnes wood chip trial.

[0236] The components of radiata pine extractives that cause pitchaccumulations are not known. Some suspect it might be the fatty acidsand/or resin acids that cause pitch to start to accumulate. It does notappear to be the overall total extractives or the triglyceride content,but this has not been proven.

[0237] From Table 11 there are the following conclusions (following fromthe statistical analysis of data provided above):

[0238] Lab Results: Total Extractives (All Lab Results are 4 Week,Fungal Incubation on Chips)

[0239] 1. In the lab, the uninoculated chips of the mill (called frozencontrol as these chips were maintained frozen until the extractives wererun on them) had sing total extractives of between 1.97% of oven dryweight of wood. Extractives of 1.97% may not seem much, but considerthat of 1000 tonnes wood chips processed per day, it could represent19.7 tonnes of extractive ‘pitch’ (if the sulfite cook process extractedlike the MTBE solvent.)

[0240] 2. In the lab, the best fungal reduction of overall extractiveswas to about 0.56% using OF 13 either grown freshly in peptone or asproduced in the powder form for the mill trial. That represents morethan two-thirds reduction of overall extractives by the fungus OF13. Thebest reduction for OF40 of overall extractives in the lab was reductionby little more than a half of overall extractives by the fungus.

[0241] Lab Results Fatty Acids and Resin Acids

[0242] 3. The best reduction of % fatty acids by fungi was OF40 at 10times inocula in YM, or OF13 at 1 times inocula in peptone; 0.14%starting control fatty acids were reduced in each case to 0.02%(reduction of 86%).

[0243] 4. The best reduction of % resin acids by fungi was OF13 (withseveral different doses and medium) and the OF40 and OF13 powderinocula; 0.67% starting control resin acids were reduced to 0.14%(reduction of 80%).

[0244] 5. The best reduction of % triglycerides by fungi was achieved byboth OF13 and OF40 and, 0.15% starting control triglycerides werereduced to 0.01% (reduction of 94%).

[0245] With both OF40 and OF13 powder, the 1× dosage used in the lab,same dosage as used in the mill, was better in total extractives, fattyacid, resin acid and triglycerides decrease than the 10 times, orone-tenth dosage.

[0246] Mill Results; Extractives OF 40 (Mill Results are Generally 10Weeks Fungal Incubation on Chips)

[0247] For OF40, the 0 week chips had total extractives of 1.94% andafter 2 weeks the total extractives had risen to 2.39%. Since this typeof shift in total extractives makes it difficult to try to analyse whatwas happening, it is best to look at FIG. 2 as the overall trend in theextractives of the chips as a function of incubation time in the chippiles. In FIG. 2 it is clear that first, there was reduction of the fourextractive parameters, and second the greatest reduction was after 10weeks than at any other time point, though % MBTE Total extractives and% triglycerides have decreased to their lowest levels by 8 weeks.

[0248] For the chips inoculated with both OF40 & OF13, see FIG. 3, itappears at 6 weeks that there was a peak of “resin” extractives. Overallagain the numbers decrease by the time 10 weeks incubation is achieved,but not as much reduced as with OF13 alone, though very close.

[0249] As the data of the OF13 will not be completed until end of Marchwe have to wait until then to understand by these parameters what is themost effective fungal inoculation. Again the caution is that no oneknows the extractive parameters that cause pitch shutdowns and they maynot be one of the four criteria tested in this analysis.

[0250] The pulps resulting from the OF40 and OF40& OF13 combinedinoculated and incubated chips were analysed also for extractives, givenin Table 11 and graphed in FIG. 4 and 5.

[0251] From this data, the OF40 pulp at the Chemiwasher stage stillcontains a significant amount of extractives The chips of OF40 at 10weeks had 1.16% MBTE total extractives, and after the sulfite cook, thepulp at the Chemiwasher has 0.92% MBTE—in other words the cook onlyremoved 20% of the total extractives. This pulp mainly contained resinacids, (20% less resin acids than were in the chips) and the fatty acidsand triglycerides in the pulps were about the same amounts as present inthe chips, but one-seventh and one-twentieth the amounts respectively asthe amount of resin acids present in the pulps. Resin acid is a biggercomponent of extractives in chemiwasher pulp than anything else wetested. Note that the components of resin acids, fatty acids andtriglycerides are only about half of the total extractives—the otherhalf is not identified.

[0252] The data of the pulps resulting from the OF40& OF13 combinedinoculated chips follows the same trends as the OF 40 pulps, thoughthere is 7% more total extractives in the OF40& OF13 chemiwasher pulpand 11% less resin acid in the OF40 & OF 13 chemiwasher pulp than theOF40 chemiwasher pulp. Again, it looks like resin acids are more presentin the chemiwasher pulp than the other components identified, and thereis little extractive in the other pulps.

[0253] The analysis of the OF13 & Of40 pitch deposit showed that 89% ofthe pitch deposit was MBTE solvent exactable. Of the extractables wehave identified only about one-third of the components. The depositcontains three times the amount of fatty acid as resin acids, and notmuch triglycerides. That deposited pitch has 22% fatty acids versus 7%resin acids.

[0254] Lipase Assay on Trial Organisms

[0255] Fungi, in order to colonise wood effectively, have to be able tosecrete specific enzymes to readily metabolise the essential compounds.One such extracellular enzyme, lipase is particularly important in thisstudy to understand better the mechanism of resin degradation. Lipasecatalyses the hydrolysis of triglycerides to glycerol, fatty acids andresin acids. Below is a brief outline of a lipase assay which usesp-nitrophenyl palmitate (PNP) as a substrate. An enzyme unit is thatamount of enzyme that will cause release of 1 μmole of PNP per minuteunder assay conditions.

[0256] The fungi used in this assay were isolated off the 8 weeksincubated wood chips, inoculated with either OF40, OF40&13 or OF13.Overnight cultures of the fungi were standardised to 10⁷ cells/ml andinoculated into Peptone and YM media

[0257] Assay

[0258] Substrate p-nitrophenyl palmitate; Assayed at 40° C. for 20minutes.

[0259] Cooled on ice and reaction stopped with 0.1 M Na₂CO₃+10% Triton.

[0260] Absorbance read at 400 nm. Activity: ΔA⁴⁰⁰/0.011×10/Time=mU/mlLipase Activity (mU/ml/10⁷ cells) Organism Peptone Media YM Media 8weeks samples 467 131 (Mixture of O. floc 40 & O. floc. 13) O. floccosum40 475 107 O. floccosum 13 365 62 

[0261] It can be concluded that these strains of Ophiostoma floccosum,as isolated of the wood chips, produced a very significant amount oflipase enzyme, This explains its ability to invade sapwood soeffectively and to be able to use resin as a nutrient source. TheUniversity of Waikato lab is now looking for other resin degradingenzymes to remove more of the resin components.

Example 6

[0262] Results

[0263] 1. In the lab, looking at the statistics of the extractivesdecrease, strain, media and the interaction between them did notsignificantly affected to % MTBE

[0264] 2. Use of fungi at the mill went smoothly with fungi inoculasprayed evenly to inoculate wood chips as conveyed by the system asevidenced by the good growth of the albino fungi on chips assayed from 2weeks chip pile incubation to 10 weeks incubation.

[0265] 3. The mill had no shutdowns when using the fungal inoculatedchips with less seasoning than routinely done.

[0266] References

[0267] Farrell, R. L., Blanchette, R. A., Brush, T. S., Hadar, Y.,Iverson, S., Krisa K., Wedler, P. A., Zimmerman, W. (1993). Cartpip TM:A biopulping product for control of pitch and resin acid problems inpulp mills. J. Biotechnol. 30: 115-122.

[0268] Farrell R. L., Blanchette, R. A., Brush, T. S., Gysin, B., Hadar,Y., Perollaz, J. J., Wendler, P. A., and Zimmerman W. “Cartapip®” ABiopulping Product for Control of Pitch and Resin Acid problems in PulpMills in Biotechnology in the Pulp and Paper Industry, Kyoto, JapanEditor: M. Kuwahara and M. Sbimada, Uni Publishers Co., Ltd., Tokyo,Japan 1992, p. 27-32.

[0269] Hoffmann, G. C., Brush, T. S., Farrell, R. L., “A BiopulpingProduct for Control of Pitch and Resin Acid Problems in Pulp and PaperProducts”, Naval Stores Review, 102(3) 10-12 (1992).

[0270] Kay, S. J., Farrell, R. L., Hadar, E., Hadar, Y., Blanchette, R.A., Harrington, T. C. (1997). Sapstain in New Zealand—the cause and apotential anti-sapstain solution. Proceedings on the 11th BiennialConference of the Australasian Plant Pathology Society, pp.21.

[0271] Wendler, P. A., Brush, T. S., Iverson, S., Krisa, K., Zimmerman,W., and Farrell, R. L., “Biological Control of Pitch Problems in PaperMills”, Kemia Kemi 19, 262-264 (1992).

[0272] White-McDougall, W. J., Blanchette, R. A., Farrell, R. L. (1998).Biological control of the stain fungi on Populus tremuloides usingselected Ophiostoma isolates. Hozforschung, 52(3), 234-240.

[0273] Behrendt, C. J., R. A. Blanchette and R. L. Farrell 1995. Anintegrated approach, using biological and chemical control, to preventblue stain in pine logs. Can J. Bot. 73:613-619.

[0274] Behrendt C J, Blanchette, R A and Farrell, R L. (1995) BiologicalControl of Blue Stain Fungi in Wood: Investigation of FungalInteractions in the Laboratory and Field. Phytopathology 85, 92-97.

[0275] Zimmerman, W. C., Blanchette, R. A., Bumes, T. A., Farrell, R. L.(1993). Melanin and perithecial development in Ophiostoma piliferum.Mycologia 87. 857-863.

[0276] Wall, M. B., Stafford, G., Noel, Y., Fritz, A., Iverson, S.,Farrell, R. (1995) Treatment with O. piliferum Improves Chemical PulpingEfficiency; 6th International Conference on Biotechnology in the Pulpand Paper Industry

1. An inoculum for wood comprising or including one or more forms of afungal culture of the species Ophiostoma floccosum and/or Ophiostomapiceae and/or Ophiosotoma pluruanulatum effective to reduce the amountof colour staining caused by wood staining fungi.
 2. An inoculum forwood as claimed in claim 1 wherein the fungal culture forms arebiologically pure.
 3. An inoculum for wood as claimed in claim 1 whereinthe fungal culture is selected from the following strains: Ophiosotomafloccosum F71, Ophiosotoma floccosum F80, Ophiosotoma floccosum F93,Ophiosotoma floccosum F40, Ophiosotoma floccosum F13, Ophiostoma piceaeOPC 703, Ophiostoma piceae OPC 422, Ophiostoma piceae OPC 580,Ophiostoma piceae OPC 194, Ophiostoma pluruanulatum 3410, Ophiostomapluruanulatum 7073, Ophiostoma pluruanulatum 5040, Ophiostomapluruanulatum 4650
 4. An inoculum for wood as claimed in claim 1 whereinthe wood source may be wood chips and the fungus or fungi may be appliedby spraying the wood chips.
 5. An inoculum for wood as claimed in claim1 wherein the wood source may be logs to be cut into structural wood,and inoculation includes inoculating at least one end of the logs, andpreferably all around the logs' surfaces.
 6. An inoculum for wood asclaimed in claim 1 therein the wood source may be structural wood andinoculation includes inoculating at least 60% of the surface area of thestructural wood.
 7. An inoculum for wood as claimed in claim 1 whereinthe wood source may be structural wood and inoculation includesinoculating at least 60% of the surface area of the structural wood,which may then be later made into chips or a subsequent fibre product.8. An inoculum for wood as claimed in claim 1 wherein the wood to beinoculated may be a conifer such as but not limited to Radiata pine,Douglas fir or Loblolly pine, and it may also be a hardwood includingbut not limited to eucalyptus, oak, poplar, or aspen.
 9. A biologicallypure culture of a strain of Ophiostoma floccosum having all of theidentifying characteristics of one of the fungi of stains F40, F13, F71,F80 and F93.
 10. A biologically pure culture of a strain of Ophiostomapiceae having all of the identifying characteristics of one of the fungiof strains OPC 703, OPC 422, OPC 580 and OPC
 194. 11. A biologicallypure culture of a strain of Ophiostoma pluruanulatum having all of theidentifying characteristics of one of the fungi of strains 3410, 7073,5040 and
 4650. 12. A method of reducing the amount of colour stainingcaused by wood staining fungi in and/or on a wood source which comprisesor includes inoculating at least a portion of the wood source with aneffective amount of at least one fungus selected from the groupconsisting of the species Ophiosotoma floccosum, Ophiosotoma piceae andOphiostoma pluruanulatum, which is capable of reducing the amount ofcolour staining caused by the wood staining fungi.
 13. A method ofreducing the amount of colour staining as claimed in claim 12 whereinthe fungi may be selected from the following stains: Ophiosotomafloccosum F13, Ophiosotoma floccosum F40, Ophiosotoma floccosum F71,Ophiosotoma floccosum F80, Ophiosotoma floccosum F93, Ophiostoma piceaeOPC 703, Ophiostoma piceae OPC 422, Ophiostoma piceae OPC 580,Ophiostoma piceae OPC 194, Ophiostoma pluruanulatum 3410, Ophiostomapluruanulatum 7073, Ophiostoma pluruanulatum 5040, Ophiostomapluruanulatum 4650
 14. A method of reducing the amount of colourstaining as claimed in claim 12 wherein the wood source may be woodchips and the fungus or fungi may be applied by spraying the wood chips.15. A method of reducing the amount of colour staining as claimed inclaim 12 wherein the wood source may be logs to be cut into structuralwood, and inoculation includes inoculating at least one end of the logs,and preferably all around the logs' surfaces.
 16. A method of reducingthe amount of colour staining as claimed in claim 12 wherein the woodsource may be structural wood and inoculation includes inoculating atleast 60% of the surface area of the structural wood.
 17. A method ofreducing the amount of colour staining as claimed in claim 12 whereinthe wood source may derive from a conifer such as but not limited to,Radiata pine, Douglas fir or Loblolly pine and it may also be a hardwoodincluding but not limited to eucalyptus, oak, poplar, or aspen.
 18. Aninoculum for wood comprising or including one or more forms of a fungalculture of the species Ophiostoma floccosum, and/or Ophiostoma piceaeand/or Ophiosotoma pluruanulatum effective in reducing the pitch contentof wood.
 19. An inoculum for wood as claimed in claim 18 wherein thefungal culture forms are biologically pure.
 20. An inoculum for wood asclaimed in claim 18 wherein the fungi may be selected from the followingstrains: Ophiosotoma floccosum F40 Ophiosotoma floccosum F13 Ophiosotomapiceae OPC 422 Ophiosotoma piceae OPC 580 Ophiosotoma piceae OPC 194 21.An inoculum for wood as claimed in claim 18 wherein the wood source maybe wood chips and the fungus or fungi may be applied by spraying thewood chips.
 22. An inoculum for wood as claimed in claim 18 wherein thewood source may be logs to be cut into structural wood, and inoculationincludes inoculating at least one end of the logs.
 23. An inoculum forwood as claimed in claim 18 wherein the wood source may be structuralwood and inoculation includes inoculating at least 60% of the surfacearea of the structural wood.
 24. An inoculum for wood as claimed inclaim 18 wherein wood to be inoculated may be but is not limited to aconifer such as Radiata pine, Douglas fir or Loblolly pine and it mayalso be a hardwood including but not limited to eucalyptus, oak, poplar,or aspen.
 25. A biologically pure culture of a strain of Ophiostomafloccosum having all of the identifying characteristics of one of thefungi of stains F40 and F13.
 26. A biologically pure culture of a strainof Ophiostoma piceae having all of the identifying characteristics ofone of the fungi of strain OPC 422, OPC 580, OPC
 194. 27. A method forreducing the pitch content in a wood source, and/or improving brightnessin chemical pulp resulting from the albino treatment of wood, and/orimproving pulping efficiency such that lower kappa numbers were achievedwith treatment of the wood with the albino strains, which comprises orincludes inoculating at least a portion of the wood source with aneffective amount of at least one fungus selected from the groupconsisting of the species Ophiosotoma floccosum, Ophiosotoma piceae andOphiostoma pluruanulatum, which is capable of reducing the pitchcontent.
 28. A method as claimed in claim 27 wherein the fungi may beselected from the following strains: Ophiosotoma floccosum F40Ophiosotoma floccosum F13 Ophiostoma piceae OPC 422 Ophiostoma piceaeOPC 580 Ophiostoma piceae OPC 194
 29. A milked as claimed in claim 27wherein the method further includes the step of maintaining theinoculated wood source under conditions which allow fungal growth fromthe inoculation for a term sufficient to effect a reduction of the pitchcontent of the wood source by such inoculated fungal growth.
 30. Amethod as claimed in claim 27 wherein the wood source may be pulpwood,or unsterilised pulpwood, or unsterilised refined pulpwood.
 31. A methodas claimed in claim 27 wherein the wood source may be wood chips and thefungus or fungi is applied by spraying the wood chips.
 32. A method asclaimed in claim 27 wherein the wood source may be debarked orundebarked timber or logs.
 33. A method as claimed in claim 27 whereinthe wood source may derive from but not limited to a conifer such asRadiata pine, Douglas fir or Loblolly pine and it may also be a hardwoodincluding but not limited to eucalyptus, oak, poplar, or aspen.
 34. Amethod as claimed in claim 27 wherein the method may include using orapplying more than one fungal strain.
 35. A biologically pure culture ofa strain of Ophiostoma floccosum having all of the identifyingcharacteristics of one of the fungi of AGAL Accession NumbersNM00/12246, NM00/12247, NM00/12490, NM00/12491 or NM00/12492.
 36. Abiologically pure culture of a stain of Ophiostoma piceae having all ofthe identifying characteristics of one of the fungi of AGAL AccessionNumbers NM00/12248, NM00/12249, NM00/12250 or NM00/12489.
 37. Abiologically pure culture of a strain of Ophiostoma pluruanulatum havingall of the identifying characteristics of one of the fungi of AGALAccession Numbers NM00/12251, NM00/12252,NM00/12253 or NM00/12493. TABLE11 Extractive Analysis of Lab and Mill Research with O. floccosumstrains OF40 and OF13 % Fatty acids % Resin acids Total on OD on %Triglicerides on Treatment % MTBE Wood OD wood OD Wood Frozen control1.97 0.14 0.67 0.16 F13 1/10 YM 0.71 0.08 0.13 0.03 F13 × YM 1.09 0.080.31 0.02 F13 10 × YM 1.32 0.09 0.42 0.01 F40 ′1/10 × YM 1.88 0.12 0.770.1 F40 × YM 1.14 0.09 0.24 0.03 F40 10 × YM 1 0.02 0.41 0.02 F13 + F40× YM 1.75 0.14 0.65 0.09 F13 1/10 × Peptone 1.15 0.03 0.5 0.03 F13 ×peptone 0.59 0.02 0.14 0.01 F13 10 × peptone 0.58 0.03 0.18 0.01 F401/10 × peptone 1.04 0.07 0.37 0.07 F40 × Peptone 0.9 0.03 0.28 0.01 F4010 × peptone 1.28 0.04 0.5 0.02 F13 1/10 × powder 1.04 0.08 0.35 0.03F13 × powder 0.56 0.06 0.16 0.03 F13 10 × powder 1.59 0.1 0.47 0.04 F401/10 × powder 1.15 0.08 0.29 0.04 F40 × powder 0.91 0.08 0.20 0.03 F4010 × powder 0.97 0.1 0.19 0.05 Mix F13 + F40 1/10 0.74 0.07 0.14 0.03powder Mix F13 + F40 × 1.06 0.11 0.27 0.04 powder Mix F13 + F40 10 × 10.11 0.24 0.04 powder Mill Trial % MTBE % extractives % Fatty acids %Resin acids Triglycerides Treatment On OD wood on OD wood on OD wood onOD wood F40 0 Wk 1.94 0.09 0.94 0.31 F40 2 wk 2.39 0.15 1.23 0.21 F40 4wk 1.47 0.08 0.67 0.04 F40 6 WK 1.86 0.06 0.93 0.04 F40 8 wk 1.41 0.040.63 0.03 F40 10 wk 1.16 0.04 0.46 0.01 F40 pulp 0.92 0.05 0.37 0.02Chemiwasher F40 pulp Caustic 0.23 0.02 0.06 0.02 F40 pulp TWWP 0.1* 0.00.01 0.01 F40 pulp bleached 0.1* 0.01 0.01 0.01 F13 + F40 2 wk 1.71 0.10.72 0.18 F13 + F40 4 wk 1.45 0.09 0.55 0.08 F13 + F40 6 wk 2.24 0.091.09 0.06 F13 + F40 8 Wk 1.15 0.05 0.38 0.03 F13 + F40 10 wk 1.4 0.070.49 0.02.